1. Field of the Invention
The present invention relates to a macrophage stimulating protein variant wherein a cysteine residue at position 672 from the N-terminus in the amino acid sequence of macrophage stimulating protein is deleted or substituted by another amino acid residue; a DNA fragment encoding the variant; a recombinant vector including the DNA fragment; a host cell transformed with the recombinant vector; and a method for producing the macrophage stimulating protein variant by culturing the transformed host cell.
2. Description of the Related Art
Macrophage stimulating protein (hereinafter, referred to as MSP) enhances phagocytosis, stimulates chemotaxis, and induces morphological changes of, for example, murine resident peritoneal macrophages (Leonard et al., Exp. Cell. Res., 102, 434 (1976); Skeel et al., J. Exp. Med., 173, 1227 (1991)). Because MSP has the ability to activate macrophages, which belong to phagocytes, it is expected that MSP may serve as a preventive pharmaceutical agent for infectious disease.
MSP was first isolated and purified from human serum, and its partial amino acid sequence was then determined (Skeel et al., J. Exp. Med., 173, 1227 (1991); Leonard et al., U.S. Pat. No. 5,219,991).
A nucleotide sequence of cDNA independently isolated from a hepatocarcinoma cell line HepG2, encoding an amino acid sequence homologous to that of hepatocyte growth factor, which was designated as a hepatocyte growth factor-like protein (HGF-like protein), was reported (Han et al., Biochemistry, 30, 9768 (1991); Degen et al., U.S. Pat. No. 5,315,000). Thereafter, it was reported that an amino acid sequence encoded by cDNA which was cloned using the partial amino acid sequence of MSP was completely identical with that of HGF-like protein (Yoshimura et al., J. Biol. Chem., 268 15461 (1993)). Furthermore, by observing that a culture supernatant of COS cells transfected with an expression vector for the HGF-like protein-encoding cDNA had MSP activity, it was confirmed that MSP is identical with HGF-like protein.
MSP is a glycoprotein composed of 693 amino acids excluding signal sequence. MSP is secreted as a single chain inactive form (hereinafter, referred to as pro-MSP). Pro-MSP is specifically cleaved between Arg (493) and Val (494) with an enzyme such as human kallikrein and converted into a two-chain active form MSP (Wang et al., J. Biol. Chem., 269, 3436 (1994)). Native MSP purified from human serum is in a two-chain form The native MSP migrates as a single band with a molecular weight of about 70 Kd on SDS electrophoresis under non-reducing condition, while it is separated into an .alpha.-chain band with a molecular weight of about 47 Kd and a .beta.-chain band with a molecular weight of about 22 Kd under reducing condition. More specifically, the .alpha.-chain (Gln19 to Arg493) and the .beta.-chain (Val494 to Gly711) of MSP are cross-linked through a disulfide bond. It is considered that a disulfide bond is formed between Cys (468) and Cys (588) in the amino acid sequence of MSP through the comparative study with the amino acid sequences of hepatocyte growth factor and plasminogen.